One of the central post-translational control elements in eukaryotic signal transduction is the phosphorylation of the hydroxyl moiety of a kinase serine, threonine, or tyrosine. The phosphorylation state of a given protein can govern its enzyme activity, stability, protein-protein binding interactions, and cellular distribution. Phosphorylation and dephosphorylation is thus a “chemical switch” that allows the cell to transmit signals from the plasma membrane to the nucleus, and to ultimately control gene expression. Kinases are involved in the control of cell metabolism, growth, differentiation, and apoptosis. Kinase signaling mechanisms have been implicated in the onset of cancer, metabolic disorders (for example diabetes), inflammation, immune system disorders, and neurodegeneration. Certain kinases have been implicated in cell proliferation and carcinogenesis. For example, many human cancers are caused by disregulation of a normal protein (e.g., when a kinase proto-oncogene is converted to a kinase oncogene through a gene translocation).
Because kinases are key regulators they are ideal drug design targets. Inhibitors of kinases are among the most important classes of pharmaceutical compounds known. Highly selective, cell-permeable modulators of one or more individual kinases are useful for the treatment of various kinase-implicated disorders. Kinase modulating compounds are additionally useful for the systematic investigation of the cellular function of one or more kinases, and thus, provide valuable tools for the identification of various kinases of therapeutic interest.
Some kinase inhibitors, including tyrosine kinase inhibitors, inhibit T-cell proliferation, and are thus useful as immunosuppressive agents for the prevention or treatment of graft rejection following transplant surgery and for the prevention or treatment of autoimmune diseases such as rheumatoid arthritis and psoriasis.
Kinases also play a critical role in angiogenesis. Angiogenesis, the formation of new blood vessels from preexisting ones, plays a critical role in many pathological settings, including cancer, chronic inflammation, diabetic retinopathy and macular degeneration. Angiogenesis is regulated by multiple cell-signaling pathways, including pathways controlled by cellular kinases. Blocking angiogenesis, through the modulation of cell kinases, therefore, represents an effective approach to the treatment of diseases such as cancer.
The process of angiogenesis is complex, requiring the concerted actions of multiple angiogenic mediators as well as the participation of different cell types. Key angiogenesis mediators, including vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), and angiopoietin 1 and 2 (Ang1 and Ang2) that bind to their cognate receptors (VEGFRs, FGFRs and Tie1 and Tie2, respectively) expressed on endothelial cells, as well as platelet-derived growth factor (PDGF) that binds to its receptor (PDGFRs) expressed on pericytes and smooth muscle cells, have been identified. Recent studies indicate that several members of the ephrin family and their receptor Eph family are novel regulators of vascular differentiation and development that have also been implicated in the angiogenesis in tumors. Thus, inhibitors of EphB4 tyrosine kinase are desirable as anti-cancer agents and anti-angiogenesis agents.
Concurrent modulation of multiple cell-signaling pathways is also desirable for the treatment of cancer. Oncogenic signaling pathways have been shown to be mediated by kinase activity; therefore, modulating these oncogenic kinases is desirable for the treatment of cancer. Kinases that have been implicated in oncogenic signaling include, but are not limited to: EGFR, ErB2, c-Kit, PDGFRα, and Flt3.
Many of the cellular processes regulated by kinases are further regulated by Hsp90.
Hsp90 is a molecular chaperone, a class of proteins that regulates protein folding in cells. Hsp90 is a 90 kD protein that functions as a homodimer. Hsp90 regulates its own expression by sequestering the transcription factor, HSF1, under non-stress conditions. Upon heat shock, HSF1 is released from Hsp90 leading to transcription and increased synthesis of Hsp90, thereby controlling the cellular stress response.
Numerous contacts in the 190 C-terminal amino acids of the protein are responsible for dimerization of this protein. The 25 kD NH2-terminal of Hsp90 contains an ATP binding site, where ATP is bound and subsequently hydrolyzed. Thus Hsp90 is an ATPase, and has been classified as a member of the GHKL ATPase superfamily. It is believed that unfolded, or partially folded substrate proteins, also called Hsp90 client proteins, are stably bound to Hsp90 in its ATP bound state, and released upon ATP hydrolysis.
Hsp90 is an important cell cycle regulatory protein, implicated in the correct folding of multiple proteins in the mitogenic signal cascade. Hsp90 also plays a role in cyclin dependent progression through G1 and G2 and in centrosome function in mitosis. Hsp90 substrates include a number of steroid hormone receptors including the androgen receptor (AR), estrogen receptor, and glucocorticoid receptor.
Hsp90 has been specifically implicated in the proper folding of a number of tyrosine and threonine kinases. It also insures the correct folding and activity of numerous kinases involved in cell proliferation and differentiation, many of which also play roles in oncogenesis.
Hsp90 can also function as part of a multi-component complex interacting with many other co-chaperone proteins. While Hsp90 forms a multi-component complex to some extent in normal cells, nearly all Hsp90 present in cultured tumor cells has been shown to be part of a multi-component complex. A number of known oncogenic proteins that are Hsp90 substrate proteins, depend on the chaperone activity of the Hsp90 complex for correct folding. Thus Hsp90 functions as a supplier of oncogenic proteins in tumor cells. Hsp90 complex in tumor cells also exhibits higher ATPase activity than Hsp90 from non-cancerous cell lines.
Geldanamycin, a natural product, is an Hsp 90 inhibitor that binds to the ATP binding site of Hsp90 inhibiting ATP hydrolysis but not substrate protein binding. Substrate proteins that reside longer on Hsp90 when ATP hydrolysis is inhibited are ubiquinated, and subsequently degraded. Disrupting the function of the Hsp90 complex has been shown to deplete oncogenic kinases (via ubiquitin-mediated proteasomal degradation) and decrease tumor growth. The Hsp90 complex present in tumor cells exhibits much higher affinity for geldanamycin and for 17-AAG, a geldanamycin derivative, than Hsp90 in non-tumor cells. Thus inhibitors of the Hsp90 complex have the ability to convert this protein from a chaperone that insures correct protein folding of oncogenic proteins to a selective protein degradation tool.
Because of its roles in cell cycle control, cell growth, and oncogenesis the Hsp90 complex is an important target for anti-cancer therapeutics. The ability of certain Hsp90 complex inhibitors to cause this protein complex to selectively target its substrate proteins for degradation makes the Hsp90 complex an especially desirable anti-cancer target. Hsp90 is also a potential drug target for autoimmune and degenerative disease because of its role in modulating the cellular stress response.
Agents capable of modulating the Hsp90 complex, are highly desirable for the treatment of a variety of diseases and disorders, including cancer. Small molecule, non-peptide antagonists of the Hsp90 complex are of particular value for such therapies. The present invention fulfills this need, and provides further related advantages.